Three membrane proteins in the rod photoreceptors, rhodopsin, peripherin/rds and cGMP-gated channels, localize to different sub-membrane compartments of the outer segments. This differential compartmentalization is essential for phototransduction signaling, as well as the morphogenesis and maintenance of the rod outer segments. Since photoreceptor outer segments do not have machinery for protein synthesis, proteins need to be synthesized and transported from the photoreceptor inner segments by an intraflagellar transport mechanism. Deficiencies in such transport have been implicated in a broad spectrum of diseases collectively called "retinal ciliopathy". Details of how photoreceptor outer segment morphogenesis is regulated have been controversial;however increasing evidence appears to support a direct association between intraflagellar transport malfunction and retinal ciliopathy. We have developed a new method to fluorescently label newly synthesized proteins in rod photoreceptors and track their movements via confocal and two-photon microscopy in living Xenopus laevis tadpoles. By using this method, we propose: Aim 1. Elucidate the process of photoreceptor disk membrane morphogenesis.;Aim 2. Dissect the functions of signal sequences located at the C-terminal regions of photoreceptor outer segment specific proteins. In Aim 1, we will study the process of disk morphogenesis by using rhodopsin and peripherin/rds fused to a novel fluorescent protein Dendra2. Dendra2 is a photoactivatable protein which turns from green to red upon irradiation by UV or intense blue light, and is able to label newly synthesized proteins in green. In Aim 2, we will differentiate the trafficking pathways mediated by the C-terminal region of three proteins--rhodopsin, peripherin/rds and cGMP-gated channel [unreadable]- subunit. Toward this goal, we will use various Dendra2 fusion proteins and live tadpole imaging. In another aim, we will elucidate the yet unsolved and controversial role of rhodopsin in the process of outer segment morphogenesis. We propose: Aim 3. Determine the structural role of rhodopsin in the integrity of the outer segment. In this aim, we will use a newly established animal model which expresses melanopsin instead of rhodopsin to form the outer segments. We will then determine if the specific structure of rhodopsin is contributing to the integrity of the outer segments by using rhodopsin-melanopsin chimeras expressed in Xenopus rod photoreceptors. The outer segment is not observed in rhodopsin knockout mice, and rhodopsin mislocalization is observed in a broad spectrum of retinal ciliopathies such as Bardet Biedl Syndrome, Usher Syndrome, and other non-syndromic retinitis pigmentosa. Understanding the process and mechanism of outer segment morphogenesis could reveal novel pathways which involve the products of ciliopathy causative genes. PUBLIC HEALTH RELEVANCE: Light is sensed by a part of the photoreceptor neuron called the "outer segment," which disappears or gets shorter in blinding diseases called "ciliopathies". To better understand the process of ciliopathies, we study in two ways how the outer segment is formed. First, we visualize directly the process of outer segment formation by microscopy. Second, we will determine the structural role of the key protein, rhodopsin, in building the outer segment.